1
Atherosclerosis is evident in treated HIV-infected subjects with low cardiovascular risk by carotid cardiovascular magnetic resonance
Short title: Carotid CMR in treated HIV disease
Authors
Kathleen A.M. ROSE*, 2, Jaime H. VERA*, 1, Peter DRIVAS, Winston BANYA, Niall KEENAN, Dudley J. PENNELL, Alan WINSTON
1 Section of Infectious Diseases, Department of Medicine, Imperial College London, London, UK.
2Cardiovascular Biomedical Research Unit, Royal Brompton Hospital, London, UK, SW3 6NP.
3Royal Brompton Hospital, Sydney Street, London, SW3 6NP, UK.
*Joint first authors
Corresponding Author:
Dr Jaime Vera
Clinical Trials, Winston Churchill Wing, St. Mary’s Hospital
Imperial College London, Praed Street, London W2 1NY, UK
Phone/Fax: +44 203 312 1603/6123
Email:
Acknowledgements
The authors are grateful to the staff of the CMR Unit and the Cardiovascular BRU, Royal Brompton Hospital for their support with this work and the medical and administrative staff at St Mary’s Hospital who assisted with data collection.
Authors’ contributions
AW and JHV conceived the study. DJP and KR advised on the study design. KR, JHV and PD were involved in data gathering. KR analysed CMR data. KR and JHV were involved with data analysis and interpretation as well as drafting the manuscript. WB performed the statistical analysis. KR drafted the initial manuscript. All authors were involved in the critical revision of the manuscript and approved it.
Conflicts of Interest and Source of Funding
This work was supported by the National Institute of Health Research Cardiovascular Biomedical Research Unit at Royal Brompton and Harefield NHS Foundation Trust, and Imperial College London. KR received grant support from CORDA, the research charity. JHV is a recipient of the Wellcome Trust Translational Medicine and Therapeutics Fellowship and has received honoraria from Merck and Janssen Cilag, and sponsorship to attend scientific conferences from Janssen Cilag, Gilead Sciences and AbbVie. AW has received honoraria and research grants, been a consultant or investigator in clinical trials sponsored by Abbott, Boehringer Ingelheim, Bristol-Myers Squibb, Gilead Sciences, GlaxoSmithKline, Janssen Cilag, Roche, Pfizer and ViiV Healthcare. DJP is a consultant to Siemens and a director of Cardiovascular Imaging Solutions. Royal Brompton Hospital has a research collaboration agreement with Siemens. None of the other authors report any disclosures relevant to this work.
Ethics approval
Imperial College London NHS health care trust Ethics Committee
Word count for abstract and manuscript
Abstract – 247 including headings
Manuscript – 3311 including headings
ABSTRACT
Objective
Premature atherosclerosis has been observed among HIV-infected individuals with high cardiovascular risk using one-dimensional ultrasound carotid intima-media thickness (C-IMT). We evaluated the assessment of HIV-infected individuals with low traditional cardiovascular disease risk using cardiovascular magnetic resonance (CMR), which allows three-dimensional assessment of the carotid artery wall.
Methods
Carotid CMR was performed in 33 HIV-infected individuals (cases) (19 male, 14 female), and 35 HIV-negative controls (20 male, 15 female). Exclusion criteria included smoking, hypertension, hyperlipidaemia (total cholesterol/HDL ratio>5) or family history of premature atherosclerosis. Cases were stable on combination antiretroviral therapy (cART) with plasma HIV-1 RNA <50 copies/mL. Using computer modelling, the arterial wall, lumen, and total vessel volumes were calculated for a 4cm length of each carotid artery centered on the bifurcation. The wall/outer-wall ratio (W/OW), an index of vascular thickening, was compared between the groups.
Results
Cases had a median CD4 cell count of 690 cells/uL. Mean (±SD) age and 10-year Framingham coronary risk scores were similar for cases and controls (45.2±9.7years versus 46.9±11.6years and 3.97±3.9% versus 3.72±3.5%, respectively). W/OW was significantly increased in cases compared with controls (36.7% versus 32.5%, p<0.0001); this was more marked in HIV-infected females. HIV-status was significantly associated with increased W/OW after adjusting for age (p<0.0001). No significant association between antiretroviral type and W/OW was found – W/OW lowered comparing abacavir to zidovudine (p=0.038), but statistical model fits poorly.
Conclusions
In a cohort of treated HIV-infected individuals with low measurable cardiovascular risk, we have observed evidence of premature subclinical atherosclerosis.
KEYWORDS
Human immunodefiency virus, atherosclerosis, cardiovascular risk, magnetic resonance imaging, carotid intima-media thickness
INTRODUCTION
An estimated 35.3 million people are living with HIV worldwide.[1] An estimated 107,800 people in the UK were living with HIV in 2013, with one in four people living with HIV infection aged 50 years and over.[2] The introduction of effective combination anti-retroviral treatment (cART) in the mid-1990s has transformed HIV-infection from a fatal to a chronic lifelong condition in the developed world. Increasingly, this is also the case in low-to-middle income countries as access to treatment improves.1 Despite this, mortality rates in HIV-infected patients are still higher than in the general population and non-AIDS related morbidity and mortality is increasing.[3], [4] Cardiovascular disease, particularly ischaemic heart disease, is a leading cause of morbidity and mortality.3, [5] Although traditional cardiovascular risk factors are highly prevalent and accepted to play a role in HIV-associated cardiovascular disease,[6], [7] the role of long-term cART and HIV-infection itself remains controversial.
Atherosclerosis is a complex, active and progressive disease with inflammation involved at every stage. Chronic inflammatory diseases, such as rheumatoid arthritis,[8] and infections, such as Chlamydia pneumonia and cytomegalovirus,[9] have been shown to be associated with excess and premature cardiovascular risk. Assaults to the endothelium result in repair via up-regulation of innate and adaptive immune systems.[10] If the endothelial insult is repeated or continuous, the inflammatory process is continued, amplified and becomes maladaptive, resulting in intimal proliferation[11] and eventually in atheroma. HIV-infection causes chronic inflammation with persistently increased inflammatory markers.[12] These increase with increasing viraemia[13], [14] and predict mortality.[15] HIV-infection is associated with raised markers of endothelial activation including VCAM-1, P-selectin and MCP-1, which decrease but may not normalise with antiretroviral treatment.14 Immune dysfunction may also contribute to the increased risk for atherosclerosis in HIV-infected individuals. Relative risk of ischaemic heart disease among patients with a CD4+ cell count ≤200 cells/uL was found to be greater than in those with a cell count >200 cells/uL at antiretroviral therapy initiation.[16] Activated T-lymphocytes in HIV- infected individuals have been found to be associated with subclinical carotid artery disease.[17]
Carotid artery intima-media thickness (C-IMT) assessed with B-mode ultrasound has been shown to be predictive of future cardiovascular events in HIV-uninfected individuals.[18], [19], [20] C-IMT has been used in numerous studies to assess for the presence and rate of progression of subclinical atherosclerosis in HIV-infected individuals.[21], [22] Findings have been conflicting due to variation in study design and ultrasound methodology. The presence of confounding variables, such as a high burden of traditional cardiovascular risk factors in the HIV-infected groups, and exposure to antiretroviral therapy, has made the effect of HIV-infection itself hard to ascertain.
Carotid vessel wall imaging by cardiovascular magnetic resonance (CMR) can overcome many of the limitations of C-IMT, which include one-dimensionality, variability of measurement site, and near field artefacts. It can be performed with constant resolution along the length of the artery and combined into a three-dimensional model giving the wall volume for the length of artery studied. It has been shown to correlate well with measurements of C-IMT.[23] Reproducibility is good with interstudy coefficients of variation of 4.4%[24], allowing for a greatly reduced sample size in clinical studies.
We report the first study using CMR to assess carotid wall thickness and determine the level of subclinical atherosclerosis in HIV-infected individuals with low cardiovascular risk, compared to a low cardiovascular risk, HIV-uninfected cohort.
METHODS
HIV- infected individuals (n=33) were recruited from the outpatient HIV unit at St Mary’s Hospital, Imperial Healthcare NHS trust, London, United Kingdom. Inclusion criteria included chronic HIV-infection (HIV antibody positive for at least 2 years), male or female gender, 20 to 70 years old, stable on cART and plasma HIV RNA<50 copies/mL (Quantiplex assayTM, Bayer, Emeryville, CA, USA). Exclusion criteria were current or previous history of cardiovascular disease or positive family history of premature vascular disease, current or previous history of major modifiable risk factors for atherosclerosis (current or former smoking, hypertension, hyperlipidaemia, diabetes), Framingham cardiovascular risk of more than 10% or DAD risk of more than 5%, taking any cardiovascular medication (e.g. anti-platelet, antihypertensive, lipid-lowering medications), current alcohol abuse or recreational drug use of less than 6 months, or contraindication to CMR.. The study was approved by the Imperial College London NHS health care trust Ethics Committee (reference number: 11/LO/1059) and all subjects provided written informed consent prior to enrolment. A control group comprising a historic cohort of HIV-uninfected healthy subjects (n=35) with no known cardiovascular disease, no current or previous history of major modifiable risk factors for atherosclerosis and low cardiovascular risk scores (<10%), matched for age, gender and where possible, ethnicity (self-reported for both groups) was used.[25] CMR was performed at the Cardiovascular Biomedical Research Unit, Royal Brompton Hospital, London, United Kingdom. A single experienced investigator performed all HIV-infected subject analyses, blinded to all parameters other than HIV-status. All the CMR images were analysed using dedicated software (Atheroma Tools, a plug-in of CMR tools, Cardiovascular Imaging Solutions, London, UK).
Demographics and Blood Test
A detailed assessment including age, height, weight, BMI, ethnicity as well as HIV-disease history and assessment of cardiac risk factors including family history of coronary artery disease and Framingham cardiovascular risk scoring was carried out at a screening visit. Blood pressure, ECG and blood tests (CD4 lymphocyte count, plasma HIV RNA, fasting lipids, glucose, electrolytes, urea and creatinine) were also performed. Subjects who reported no cardiovascular risk factors but were found to have elevated blood pressure (>140/90 mm Hg), elevated total cholesterol to HDL ratio (>5), triglycerides or glucose, or abnormal ECG were excluded from the study and referred to their physician for further management.
CMR
Carotid CMR was performed using a 3.0 T scanner (Siemens Skyra, Erlangen, Germany) and a purpose-built bilateral four-channel phased-array carotid surface coil (Machnet BV, Eelde, the Netherlands), with the head and neck immobilized. A contiguous stack of high-resolution T1-weighted fast spin echo images, centred on the carotid bifurcation bilaterally, was acquired approximately perpendicular to the longitudinal axis of both common carotid arteries. Slice thickness was 2 mm, and 20 contiguous slices were acquired for each side, giving 40 mm of longitudinal coverage per artery. The coverage of common carotid and internal carotid artery on the left and right sides was identical at 20 mm above and below the bifurcation. Typical sequence parameters for T1-weighted fast spin echo images were: field of view (FOV) read 110 mm, FOV phase 100%, TE 11ms, echo train length 9, readout time 90ms, bandwidth 230 Hz/pixel, 3 averages, pixel size .43mmx0.43mm (interpolated to 0.21mmx0.21mm), ECG-gated to each cardiac cycle with end-diastolic triggering. Dark blood preparation was used with the inversion time (TI) determined by average R-R interval.
The internal and external carotid artery surfaces were manually traced giving the luminal area and the total vessel area for each slice. Where flow suppression was incomplete, sufficient separation between the flow signal and the vessel wall allowed them to be distinguished. This was assisted by 2D semi-automated modelling by the analysis software. Using the slice thickness (2mm), the lumen and wall volume was automatically calculated for each slice and summated to create a 3D model from which lumen volume, wall volume and total vessel volume were derived. The total wall volume was then expressed as a percentage of the total vessel volume (wall/outer wall or W/OW ratio – an index of vascular thickening). A cine image perpendicular to the common carotid artery was manually contoured at end diastole and end systole for each side, and the percentage distensibility calculated.
The historic non-HIV infected cohort had been previously scanned on a 1.5T scanner using a similar protocol to produce a contiguous stack of high-resolution T1-weighted images. These had been analysed using the same standard methods as employed in this study.25 Previously, measures of carotid wall volume have been compared between 1.5T and 3.0T magnetic field strengths with no significant differences reported.[26]
Sample Size
In a previous study by Keenan,25 the age-group with the highest change in wall volume (60 to 69 years old) showed a mean wall volume of 1083mm3 with an estimated standard deviation (SD) of 189. Assuming an increase of 25% in wall volume among HIV-infected individuals a sample size of 22 patients (11 cases and 11 controls) would be required, to detect differences between groups. We aimed to recruit an age and gender matched sample of 5 subjects per age group (20 to 39 years, 40 to 49 years, 50+ years) per sex.
Statistical Analyses
Categorical data were presented as number (percentage) and comparisons undertaken using the chi-squared or Fishers Exact test. Numeric data were presented as a mean (SD) or 95% confidence interval if the data were normally distributed and analysed using a 2-sample independent t-test. Where data were not normally distributed, median (IQR) was presented and comparisons done using the Mann-Whitney (Wilcoxon rank-sum) test. Linear regression analysis was performed to assess the strength of association between measured variables (W/OW, carotid artery wall volume, carotid artery lumen volume, total carotid artery volume) and age. The association of demographic and HIV-specific parameters with W/OW was analysed using linear regression.
Results
Patient Characteristics
Demographics, blood pressure and laboratory values, and coronary risk scores in HIV-infected subjects and age- and sex-matched control subjects are shown in Table 1. Age, gender distribution, total cholesterol levels, blood pressure, BSA, age and 10-year coronary risk were similar between both groups. All participants had never smoked and had no history of diabetes, hypertension or previous vascular disease.
HIV-infected subjects had a mean age of 45.2±9.7 years. Mean duration of known HIV-infection was 8.8±4.4 years. Current HIV RNA was <50 copies in all subjects. All subjects were stable on cART with a median duration of treatment of 7 years (2-21 years); 24 were on a non-nucleoside reverse transcriptase inhibitors (NNRTI) based regimen and 9 on a protease inhibitor (PI) based regimen. All subjects were receiving an NRTI backbone that comprised of tenofovir, abacavir and zidovudine in 24, 6 and 3 subjects, respectively. Of those on a PI based regimen, 5 were on darunavir, 3 were on atazanavir and 1 was on lopinavir. All patients on PIs were on a boosted combination with ritonavir.